Elisa Rabellotti

Polar body morphology and spindle imaging as predictors of oocyte quality

It has been suggested that first polar body (PBI) morphology reflects oocyte competence. Oocytes with an intact normal-sized PBI have been described as generating better day 2 embryos, higher blastocyst yield, and increased pregnancy and implantation rates. In other studies, PBI morphology was found to be unrelated to fertilization rate, embryo quality, and blastocyst formation. In a prospective analysis, the predictive value of the PBI was investigated by comparing the development of oocytes retrieved from intracytoplasmic sperm injection patients and displaying different PBI morphology, classified according to the following characteristics: normal size and smooth surface (I), fragmented (II), rough surface (III), or large size (IV). Fertilization rates were 59, 57, 64 and 60% respectively. No significant differences were found between the various groups. The proportions of high quality (grade A) day 2 embryos were also comparable among groups I-III (14, 12 and 17% respectively), while the low number of grade A embryos in group IV (two embryos) did not allow comparison with the other classes. These data do not suggest that PBI selection can contribute to identification of embryos with high developmental ability. In order to establish alternative criteria for oocyte selection, a metaphase II (MII) spindle analysis was also conducted via Polscope. In oocytes of patients of different age, spindle retardance (which reflects the high order and density of microtubules) was compared with parameters of embryo development. In aged patients, a trend was observed between low retardance and poor embryo quality, although in general the association between retardance and oocyte developmental performance did not reach statistical significance.

Oocyte cryopreservation: clinical outcome of slow-cooling protocols differing in sucrose concentration

Oocyte cryopreservation represents an important option for management of female fertility, avoiding the ethical concerns associated with embryo storage. This retrospective study evaluated the clinical outcome of two alternative slow freezing protocols involving different sucrose concentrations. From January 2004 to March 2006, spare oocytes from selected couples undergoing IVF or intracytoplasmic sperm injection were frozen using a slow-cooling protocol and thawed at a later stage. Patients were divided into two groups: group A (n = 65), whose oocytes were frozen with propane-1,2-diol (PrOH) and 0.1 mol/l sucrose; and group B (n = 66) whose oocytes were frozen with 0.3 mol/l sucrose. A total of 543 oocytes were thawed in group A and 601 in group B, achieving a survival rate of 24.3 and 71.2% respectively. Whilst fertilization rate (53.5 and 80.4% respectively) was higher in group B, enhanced results for group A were achieved over all (implantation rate per transferred embryos 12.2 versus 5.7%; pregnancy rate per transfer 16.7 versus 9.5%). Normal births and ongoing pregnancies have occurred in both groups. Although in slow-cooling methods higher sucrose concentration in the freezing mixture allows higher post-thaw survival and fertilization rates, overall this did not coincide with an improved clinical outcome.

Pathologic findings in hysteroscopy before in vitro fertilization-embryo transfer (IVF-ET)

Background. The aim of this study was to evaluate hysteroscopy routinely performed prior to in vitro fertilization-embryo transfer (IVF-ET). Methods. We analyzed in a prospective study 300 patients who underwent hysteroscopy before the first IVF-ET cycle. We analyzed then in a retrospective manner 300 patients who did not perform hysteroscopy. Results. One-hundred-and-eighty (60%) hysteroscopies were normal but 120 (40%) revealed an unsuspected intrauterine abnormality. We did not find statistically significant differences between patients with normal or abnormal hysteroscopy in any characteristic. We found a statistically significant difference in pregnancy rate between women who performed hysteroscopy before IVF-ET cycle and in women who did not perform it. Conclusions. Hysteroscopy, as a routine examination, should be performed before the first IVF-ET cycle in all patients.

Objective evaluation of the viability of cryopreserved oocytes

Recent studies of fundamental cryobiology, empirical observations and more systematic clinical experiences have generated a renewed interest in oocyte cryopreservation. Poor survival rate has long been the limiting factor which has prevented widespread adoption of oocyte storage. Slow-cooling and vitrification protocols developed in the last few years have apparently solved this problem, ensuring high recovery of viable oocytes from liquid nitrogen storage. However, the definition of oocyte viability appears rather vague. In fact, post-storage survival as assessed on morphological criteria, indicated by the absence of overt cell degeneration, is not necessarily synonymous with viability. Despite its sensitivity to low temperatures, the meiotic spindle can be preserved after cryopreservation and its constitution after thawing can be monitored non-invasively through polarized light microscopy. Assessment of oocyte cryopreservation via clinical parameters is a daunting task. Most studies are small and difficult to interpret because of confounding factors, such as age, patient selection and quality and strategy of use of the cryopreserved material. Some progress has been made, however, as suggested by recent experiences in which the implantation efficiency of embryos produced from thawed oocytes approaches that reported using cryopreserved embryos directly.

Epigenetic analysis of the critical region I for premature ovarian failure: demonstration of a highly heterochromatic domain on the long arm of the mammalian X chromosome

Background: X chromosome rearrangements defined a critical region for premature ovarian failure (POF) that extended for >15 Mb in Xq. It has been shown previously that the region could be divided into two functionally distinct portions and suggested that balanced translocations interrupting its proximal part, critical region 1 (CR1), could be responsible for POF through downregulation of ovary expressed autosomal genes translocated to the X chromosome. Results and conclusion: This study reports that such position effect can indeed be demonstrated by analysis of breakpoint regions in somatic cells of POF patients and by the finding that CR1 has a highly heterochromatic organisation, very different from that of the euchromatic autosomal regions involved in the rearrangements. The chromatin organisation of the POF CR1 is likely to be responsible for the epigenetic modifications observed in POF patients. The characteristics of CR1 and its downregulation in oocytes may very well explain its role in POF and the frequency of the POF phenotype in chromosomal rearrangements involving Xq. This study also demonstrates a large and evolutionary conserved domain of the long arm of the X chromosome, largely corresponding to CR1, that may have structural or functional roles, in oocyte maturation or in X chromosome inactivation.

Fresh blastocyst transfer as a clinical approach to overcome the detrimental effect of progesterone elevation at hCG triggering: a strategy in the context of the Italian law.

OBJECTIVE To retrospectively evaluate whether fresh day-5 embryo transfer could overcomes the detrimental effect of subtle progesterone elevations at hCG administration on pregnancy outcomes in women undergoing ovarian hyperstimulation for IVF/ICSI cycles. STUDY DESIGN Retrospective study of 204 infertile patients aged 23-44 years who underwent IVF/ICSI treatment and fresh blastocyst transfer under the Italian law (embryos cryopreservation cannot be planned in advance). Women were divided into those with a progesterone level <1.5 ng/ml and those with a progesterone concentration ≥ 1.5 ng/ml at hCG triggering. The clinical pregnancy rate (CPR) after blastocyst transfer was the primary outcome. RESULTS Age, body mass index (BMI), antral follicle count, anti-Mullerian hormone (AMH) and FSH values, mean number of stimulation days, ratio of GnRH agonist and antagonist cycles and total dose of gonadotrophins administered did not differ between the two groups. Serum estradiol and number of retrieved oocytes were significantly increased in the group with elevated progesterone and a significantly higher number of oocytes was used in this group. Fertilization rate, percentage of top quality embryos, and number of transferred blastocysts were similar in the two groups. The CPR was significantly higher in women with progesterone levels <1.5 ng/ml at hCG (50%) compared with women with progesterone concentration ≥ 1.5 ng/ml (33.3%) (odds ratio = 2.00, 95% confidence interval 1.07-3.75). CONCLUSIONS A fresh blastocyst transfer does not completely overcome the detrimental effect of progesterone rise at hCG on IVF/ICSI pregnancy outcomes.

Consecutive cycles in in vitro fertilization-embryo transfer

Background. The decline of female fertility with advancing age is well documented. The aim of this study was to compare the ovarian performance after repeated ovarian stimulation cycles in women of different ages. Methods. Four hundred patients who started at least three in vitro fertilization (IVF) cycles during the 5-year period between 1998 and 2002 were identified. The patients were divided into four groups: the 25–30 age group (n = 90), the 31–35 age group (n = 150), the 36–40 age group (n = 110) and the 41–45 age group (n = 50). Results. Comparing subsequent cycles versus the first treatment cycle we found a statistically significantly increased number of ampules of recombinant follicle stimulating hormone (rFSH) needed to reach follicles maturation (p < 0.001). The number of ampules of gonadotropin required was significantly higher (p < 0.001) in the groups of advanced age compared with the groups of young women. For women in the 36–40 group and in the 41–45 group we found the number of follicles, the number of oocytes and the proportion of grade A embryos, in every cycle, were significantly lower than in the groups of young women. We compared the characteristics of ovarian stimulation and response of a single age group in different consecutive cycles. We found significant differences (p < 0.05) only in the number of ampules required. Conclusions. Maternal age adversely affected ovarian performance. During repeated IVF cycles we also noted an age-independent decline of ovarian response.

Progesterone Resistance, Aromatase, and Inflammation: The Important Relationships Between Hormones and Inflammation

Endometriosis is a complex gynecologic disorder in which alterations of specific biological processes involving both the endocrine and the immune systems have been identified. Recently, a reduced endometrial responsiveness to progesterone has been suggested as a crucial element in the pathophysiology of the disease but it is unclear whether this reduced sensitivity to the steroid is involved in the disease induction or is simply a consequence of the disease condition. Indeed, endometriosis is associated with a local and systemic inflammatory process that is strongly inter-related to the steroid activity, and both the systems mutually interact in a complex loop that strongly influences the disease development and maintenance. The link between prostaglandin E2 (PGE2), aromatase activity, and local estrogen synthesis represents a paradigm of this loop. A better understanding of the pathophysiology of this interaction should enable researchers to develop targeted therapeutics that may limit this self-supporting pathologic loop.

Vitamin D and Endometrium: A Systematic Review of a Neglected Area of Research

Growing evidence supports a role of vitamin D (VD) in reproductive health. Vitamin D receptor (VDR) is expressed in the ovary, endometrium, and myometrium. The biological actions of VD in fertility and reproductive tissues have been investigated but mainly using animal models. Conversely, the molecular data addressing the mechanisms underlying VD action in the physiologic endometrium and in endometrial pathologies are still scant. Levels of VDR expression according to the menstrual cycle are yet to be definitively clarified, possibly being lower in the proliferative compared to the secretory phase and in mid-secretory compared to early secretory phase. Endometrial tissue also expresses the enzymes involved in the metabolism of VD. The potential anti-proliferative and anti-inflammatory effects of VD for the treatment of endometriosis have been investigated in recent years. Treatment of ectopic endometrial cells with 1,25(OH)2D3 could significantly reduce cytokine-mediated inflammatory responses. An alteration of VD metabolism in terms of increased 24-hydroxylase mRNA and protein expression has been demonstrated in endometrial cancer, albeit not consistently. The effect of the active form of the vitamin as an anti-proliferative, pro-apoptotic, anti-inflammatory, and differentiation-inducing agent has been demonstrated in various endometrial cancer cell lines.